dna recombinant technology
Post on 09-Jan-2016
175 Views
Preview:
DESCRIPTION
TRANSCRIPT
DNA Recombinant Technology DNA Recombinant Technology
DNA recombinantDNA recombinant
Genetic Engineering
The manipulation of an organism endowment by introducing or eliminating specific gene
A gene of interest is inserted into another organism, enabling it to be cloned, and thus studied more effectively
Design and construction of new combinations of genes (DNA)
New combinations/arrangements of DNA
DNA cloning
Technology used in the isolation or synthesis and joining together of unlike pieces of DNA
DNA Recombinant Technology DNA Recombinant Technology
These recombinant DNA molecules can then be introduced into bacteria, yeasts, or other cells where they can replicate and
function (code for protein synthesis)
The Application of DNA Recombinant The Application of DNA Recombinant Technology Technology
Overview of Genetic Engineering
Gene of interest is isolated from appropriate organism Gene is recombined with a vector (carrier) DNA molecule Recombinant DNA is introduced into appropriate host cell Recombinant DNA is expressed at high levels in host cell
Gene product may be purified for use in treatments (antibiotics, hormones, etc.)
WhyWhy
Detailed studies of the structure and function of a Detailed studies of the structure and function of a gene at the molecular level require large quantities gene at the molecular level require large quantities
of the individual gene in pure formof the individual gene in pure form
Cloning
A collection of molecules or cells, all A collection of molecules or cells, all identical to an original molecule or cellidentical to an original molecule or cell
To "clone a gene" is to make many copies of it To "clone a gene" is to make many copies of it - for example, in a population of bacteria - for example, in a population of bacteria
Gene can be an exact copy of a natural gene Gene can be an exact copy of a natural gene Gene can be an altered version of a natural Gene can be an altered version of a natural
gene gene Recombinant DNA technology makes it Recombinant DNA technology makes it
possible possible
ToolsTools
VectorRestriction and ligation enzymes
Host Cells
VectorVector Carriers move DNA from test tubes back into Carriers move DNA from test tubes back into
cellscells Pieces of DNA that can accept, carry, and Pieces of DNA that can accept, carry, and
replicate other pieces of DNA replicate other pieces of DNA An autonomously replicating genetic element An autonomously replicating genetic element
used to carry DNA fragments into a host for used to carry DNA fragments into a host for the purpose of gene cloningthe purpose of gene cloning
1. Bacterial plasmids
2. Bacteriophages (lambda phage)
3. Viruses4. Yeast cells
Cloning vectorsVector system Host cell Insert capacity (kb)
Plasmid E. coli 0.1-10
Bacteriophage l E. coli 10-20
Cosmid E. coli 35-45
Bacteriophage P1 E. coli 80-100
BAC (bacterial artificial chromosome)
E. coli 50-300
P1 bacteriophage-derived AC
E. coli 100-300
YAC Yeast 100-2,000
Human AC Cultured human cells
>2,000
PlasmidsPlasmidsNaturally occurring extra-chromosomal
DNA
Plasmids are circular double stranded DNA Plasmids can be cleaved by restriction
enzymes, leaving sticky ends Artificial plasmids can be constructed by
linking new DNA fragments to the sticky ends of plasmid
Maximum size of insert is about 10 kb.
LambdaLambda It has a genome of about 50 kb of linear
DNA Only 37 to 52 kb DNA fragments can be
packaged into the lambda head. Insertion vectors can hold up to 7 kb of
cDNA. Its life cycle is conducive to the use as a
cloning vector The lytic cycle can be supported by only a
portion of the genes found in the lambda genome.
Lambda life cycle.
The lytic life cycle produces phage particles immediately
The lysogenic life cycle requires genes in the middle of the genome, which can be replaced
Lambda genome
Cosmid vectorsCosmid vectors
Hybrid between a lambda vector and a plasmid. It can contain 33 to 45 kb.
Bacterial Artificial Bacterial Artificial chromosomes (BAC) vectorschromosomes (BAC) vectors
These vectors are based on the E. coli F factorThese vectors are maintained at 1-2 copies per cell and can hold > 300 kb of insert DNA.
Problems are low DNA yield from host cells.
Bacteriophage P1 These vectors are like lambda and can These vectors are like lambda and can
hold up to 110 to 115 kb of DNA . hold up to 110 to 115 kb of DNA . This DNA can then be packaged by the This DNA can then be packaged by the
P1 phage protein coat.P1 phage protein coat.The use of T4 in vitro packaging systems The use of T4 in vitro packaging systems
can enable the recovery of 122 kb insertscan enable the recovery of 122 kb inserts
Yeast Artificial Chromosomes
Many DNA fragments cannot be propagated in bacterial cells.
Therefore yeast artificial chromosomes can be built with a few specific
components.1.Centromere
2.Telomere3.Autonomously replicating sequence (ARS)
Genomic DNA is ligated between two telomeres and the ligation products are transformed into yeast cells
YAC cloning system
Cloning VectorsCloning Vectors
Plasmids that can be modified to carry new genes
Plasmids useful as cloning vectors must have Plasmids useful as cloning vectors must have • a replicator (origin of replication) a replicator (origin of replication) • a selectable marker (antibiotic resistance a selectable marker (antibiotic resistance
gene) gene) • a cloning site (site where insertion of foreign a cloning site (site where insertion of foreign
DNA will not disrupt replication or inactivate DNA will not disrupt replication or inactivate essential markersessential markers
Vectors
Three important features1. Cloning site2. Ori-an origin of replication3. A selectable marker
Coli Plasmid
pBR322pBR322
The plasmid pBR322 is one of the most commonly used E.coli cloning vectors. pBR322 is 4361 bp in length and contains: (1) the replicon rep responsible for the replication of plasmid (source – plasmid pMB1); (2) rop gene coding for the
Rop protein, which promotes conversion of the unstable RNA I – RNA II complex to a stable complex and serves to decrease copy number (source –
plasmid pMB1); (3) bla gene, coding for beta-lactamase that confers resistance to ampicillin (source – transposon Tn3); (4) tet gene, encoding tetracycline
resistance protein (source – plasmid pSC101).
pUC18/19pUC18/19
pUC18 and pUC19 vectors are small, high copy number, E.coli plasmids, 2686 bp in length. They are identical except that they contain multiple cloning sites (MCS) arranged in opposite
orientations. pUC18/19 plasmids contain: (1) the pMB1 replicon rep responsible for the replication of plasmid (source – plasmid pBR322). The high copy number of pUC plasmids is a
result of the lack of the rop gene and a single point mutation in rep of pMB1; (2) bla gene, coding for beta-lactamase that confers resistance to ampicillin (source – plasmid pBR322); (3) region of E.coli operon lac containing CAP protein binding site, promoter Plac, lac repressor binding site and 5’-terminal part of the lacZ gene encoding the N-terminal fragment of beta-
galactosidase (source – M13mp18/19). This fragment, whose synthesis can be induced by IPTG, is capable of intra-allelic (alfa) complementation with a defective form of beta-galactosidase
encoded by host (mutation lacZDM15). In the presence of IPTG, bacteria synthesize both fragments of the enzyme and form blue colonies on media with X-Gal. Insertion of DNA into
the MCS located within the lacZ gene (codons 6-7 of lacZ are replaced by MCS) inactivates the N-terminal fragment of beta-galactosidase and abolishes alfa-complementation. Bacteria
carrying recombinant plasmids therefore give rise to white colonies.
Agrobacterium tumefaciensAgrobacterium tumefaciens
Genetic structure of the Octopine Ti plasmid
TL TRAux Cyt Opines
Oncogenes
Fig. 3
Binary vector system
Binary vector system
A typical plasmid vector with a A typical plasmid vector with a polylinkerpolylinker
Chimeric PlasmidsChimeric Plasmids
Named for mythological beasts with body parts from several creatures
After cleavage of a plasmid with a restriction enzyme, a foreign DNA fragment can be inserted
Ends of the plasmid/fragment are closed to form a "recombinant plasmid"
Plasmid can replicate when placed in a suitable bacterial host
Directional CloningDirectional Cloning
Often one desires to insert foreign DNA in a particular orientation
This can be done by making two
cleavages with two different restriction enzymes
Construct foreign DNA with same two restriction enzymes
Foreign DNA can only be inserted in one direction
Host Cells
Propagation of a DNA sequence must take place inside a living cell (host cells)
Eschericia coli:It provides a relatively simple and well understood
genetic environmentThe way to isolate plasmid is understood
It contains a single chromosome of approximately 5 Mbp
The genetic code is nearly universalIt replicates once every 22 minutes
It grows best with incubation at 37°Cin a culture medium that approximately the nutrient available in the
human digestive tract
Bacterial transformation
The cellular uptake and expression of DNA in a bacteria
Introduction of DNA into competent cell of bacteriaRequested element in
transformation:1. A suitable host organism in which to
insert the gene2. A self-replicating
vector to carry the gene into the host
organism3. A means of selection
for host cells that have taken up the gene
Selection of Transformant
A particularly important selective advantage offered by plasmid is antibiotic resistance gene
It encodes for proteins that disable antibiotics secreted by microorganism with which bacteria
competeAntibiotics function by several different
mechanism
Antibiotics resistance:A selectable marker that allows one to positively
identify cells that have been induced to take up plasmid DNA
Penicillin family (including ampicillin) interfere with cell
wall biosynthesisKanamycin, tetracyclin, and chloramphenicol arrest
bacterial cell growth by blocking various steps in protein synthesis
Selectable Marker Gene
Antibiotic Description
Ampicillin (Amp) Inhibits bacterial cell wall synthesis; inactivated by b-lactamase, which cleaves the b-lactam ring of amp
Kanamycin (Kan) Binds to 30S ribosomal subunit and inhibits protein synthesis; inactivated by a phosphotransferase
Neomycin (Neo) Binds to 30S ribosomal subunit and inhibits protein synthesis; inactivated by a phosphotransferase
Tetracycline (Tet) Binds to 30S ribosomal subunit and inhibits protein synthesis; tetr gene encodes a protein which prevents transport of tet into the cell
Protein expression
- Gene is inserted into plasmid- Plasmid is transformed into a host cell (E. coli)- Cell culture is prepared- Each cell contains several copies of the plasmid with gene
- Gene expression leads to the production of protein
- Protein level may reach 30% of
total cellular protein-Isolation of protein
Restriction EnzymesRestriction Enzymes
Molecular scissors which isolated from bacteria where they are used as Bacterial defense against viruses
Molecular scalpels to cut DNA in a precise and predictable manner
Enzyme produced by bacteria that typically recognize specific 4-8 base pair sequences called restriction
sites, and then cleave both DNA strands at this site
A class of endo-nucleases that cleavage DNA after recognizing a specific sequence
Members of the class of nucleases
Breaking the phosphodiester bonds that link adjacent nucleotides in DNA and
RNA molecules
EndonucleaseCleave nucleic acids at internal position
ExonucleaseProgressively digest from the ends of the nucleic acid molecules
Nuclease
Endonuclease
Type Characteristics
I Have both restriction and modification activity Cut at sites 1000 nucleotides or more away from
recognition site ATP is required
II It has only restriction site activity Its cut is predictable and consistent manner at a
site within or adjacent to restriction site It require only magnesium ion as cofactor
III Have both restriction and modification activityCut at sites closed to recognition site ATP is required
There are already more than 1200 type II enzymes isolated from prokaryotic organism
They recognize more than 130 different nucleotide sequence
They scan a DNA molecule, stopping only when it recognizes a specific sequence of nucleotides that are composed of symetrical, palindromic sequence
Palindromic sequence:The sequence read forward on one DNA strand is identical to the sequence read in the opposite direction on the complementary strand
To Avoid confusion, restriction endo-nucleases are named according to the following nomenclature
Restriction Enzymes
The first letter is the initial letter of the genus name of the organism from which the enzyme is isolated
The second and third letters are usually the initial letters of the organisms species name. It is written in italic
A fourth letter, if any, indicates a particular strain organism
Originally, roman numerals were meant to indicate the order in which enzymes, isolated from the same organisms and strain, are eluted from a chromatography column. More often, the roman numerals indicate the order of discovery
Nomenclature
NomenclatureEcoEcoRIRI E : Genus EscherichiaE : Genus Escherichia
co: Species colico: Species coli
R : Strain RY13R : Strain RY13
I : First endonuclease isolatedI : First endonuclease isolated
BamBamHIHI B : Genus BacillusB : Genus Bacillus
am: species amyloliquefaciensam: species amyloliquefaciens
H : Strain HH : Strain H
I : First endonuclease isolatedI : First endonuclease isolated
HinHindIIIdIII H : Genus HaemophilusH : Genus Haemophilus
in : species influenzaein : species influenzae
d : strain Rdd : strain Rd
III : Third endonuclease isolatedIII : Third endonuclease isolated
SpecificityEnzymeEnzyme SourceSource SequenceSequence EndEnd
BamHIBamHI Bacillus Bacillus amyloliquefaciens Hamyloliquefaciens H
GGGATCCGATCC StickStickyy
BglIIBglII Bacillus globigiiBacillus globigii AAGATCTGATCT StickStickyy
EcoRIEcoRI Escherichia coli RY13Escherichia coli RY13 GGAATTCAATTC StickStickyy
EcoRIIEcoRII Escherichia coli R245Escherichia coli R245 CCTGGCCTGG StickStickyy
HaeIIIHaeIII Haemophilus aegyptiusHaemophilus aegyptius GGGGCCCC BluntBlunt
HindIIHindII Haemophilus influenzae Haemophilus influenzae RdRd
GTPyGTPyPuACPuAC BluntBlunt
HindIIIHindIII Haemophilus influenzae Haemophilus influenzae RdRd
AAAGCTTAGCTT StickStickyy
HpaIIHpaII Haemophilus Haemophilus parainfluenzaeparainfluenzae
CCCGGCGG StickStickyy
NotINotI Nocardia otitidis-Nocardia otitidis-caviarumcaviarum
GCGCGGCCGGGCCGCC
StickStickyy
PstIPstI Providencia stuartii 164Providencia stuartii 164 CTGCACTGCAGG StickStickyy
Restriction Product
Restriction enzymesRestriction enzymes
degenerate or specific sequences
kind of ends produced (5’ or 3’ overhang (cohesive=sticky), blunt=flush)
number of nucleotides recognized (4, 6,8 base-cutters most common)
whether cleavage occurs within the recognition sequence
Restriction enzymes can be grouped by:
A restriction enzyme (A restriction enzyme (EcoEcoRI)RI)
1. 6-base cutter
4. produces a 5’ overhang (sticky end)
2. Specific palindromic sequence (5’GAATTC) 3. Cuts within the recognition sequence (type II enzyme)
Restriction enzymesRestriction enzymes
top related